LC-MS/MS method for quantification of baclofen in hair: A useful tool to assess compliance in alcohol dependent patients?

To evaluate adherence to treatment, we developed and validated a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for baclofen quantification in hair.Twenty mg was washed twice with dichloromethane, incubated in phosphate buffer (pH 5) for 10 minutes at 95°C, then extracted by liquid-liquid extraction in alkaline condition. Baclofen-d4 was used as the internal standard. This method was applied to assess compliance in4 treated alcohol-dependent patients (3 dead and one living). Blood quantification of baclofen and ethanol were performed in the 4 cases. Hair ethylglucuronide (ethanol metabolite, EtG) measurement (2x3 cm) was associated in 1 patient. Baclofen quantification in hair was validated over the range 10-5000 pg/mg. The accuracy was within 96.0%-110.9% and the precision was less than 9.3%. Baclofen segmental (3x2cm) hair concentrations found in the living patient were 4420, 4260, and 4380 pg/mg, reflecting a regular exposure over the last 6 months and suggesting patient compliance. However, the high EtG level found in this patient in the analyzed segments (225 pg/mg and 215 pg/mg) showed excessive alcohol consumption during the same period, suggesting therapeutic failure. In the 3 deceased patients, the non-segmental analysis of hair showed baclofen concentrations of 15, 545, and 2475 pg/mg. The low concentrations in the 2 first cases are compatible either with a poor compliance or to a beginning of a treatment. This is the first measurement of baclofen in hair of alcohol dependent patients. It could be used as a monitoring biomarker to assess patient's compliance.

Microscopic acute lesions after caustic exposure.

Although lesions related to chemical burns have been studied through case reports, clinical analyses and autopsy series, microscopic lesions have not yet been precisely described. Our study analyses the microscopic lesions recorded after caustic exposure in fourteen lethal and four non-lethal cases. We find that microscopic lesions after caustic exposure are various and non-specific. Moreover, the distribution of gastrointestinal lesions is inconsistent. Histological changes affect the digestive mucosa first, with the entire wall suffering damage in some cases. Multiple factors influence the pattern of lesions, including the nature of the caustic substance, the duration of contact, the amount of the substance encountering the tissue and the length of postingestion survival. The assessment of microscopic lesions, especially necrosis, can be limited by post-mortem autolysis, which quickly affects the digestive tract. Chemical pneumonia due to caustic burns is rare and, when present, typically secondary to aspiration. According to the presented findings, macroscopic examination at autopsy under- or overestimates the nature and degree of lesions. Significant complications of caustic ingestion such as chemical pneumonitis can also be found by histological analysis. Microscopic examination can be useful to rule out oesophagitis or other digestive pathologies that can mimic chemical burns.

Postmortem abdominal CT: assessing normal cadaveric modifications and pathological processes.

PURPOSE: To investigate the interest of postmortem non-enhanced computer tomography (CT) for abdominal lesions in a forensic context of suspicions death and to list the different radiological cadaveric modifications occurring normally at abdominal stage, which must be known by non forensic radiologists in case of any postmortem exam. MATERIALS AND METHODS: 30 cadavers have been submitted to a body CT-scan without injection of contrast material. CT exams were reviewed by two independent radiologists and radiological findings were compared with forensic autopsy data. RESULTS: False positive CT findings included physiological postmortem transudates misdiagnosed with intra-abdominal bleedings, and putrefaction gas misdiagnosed with gas embolism, aeroporty, aerobily, digestive parietal pneumatosis. Incidentalomas without any role in death process were also reported. False negative CT findings included small contusions, vascular thromboses, acute infarcts foci, non radio-opaque foreign bodies. Normal cadaveric modifications were due to livor mortis and putrefaction, and are seen quickly (some hours) after death. CONCLUSION: The non forensic radiologist should be familiar with the normal abdominal postmortem features in order to avoid misdiagnoses, and detect informative lesions which can help and guide the forensic practitioner or the clinical physician.

Plakophilin 2A is the dominant isoform in human heart tissue: consequences for the genetic screening of arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease in which mutations affecting Plakophilin-2 (PKP2) are the most frequently detected. However, pathogenicity of variants is not always fully determined. PKP2 encodes two isoforms, the longest (PKP2b) includes the alternatively spliced exon 6, which is routinely screened for molecular diagnosis, despite the absence of data on cardiac expression of PKP2 isoforms. OBJECTIVE: To examine the pathogenicity of PKP2 exon 6 mutations by focusing on a missense variant located in this exon. METHODS AND RESULTS: The PKP2 heterozygous p.Arg490Trp variant was identified in two unrelated ARVC probands (absent from 470 controls). In silico analysis suggested that PKP2 exon 6 is an Alu-derived sequence with very low expression level. PKP2a mRNA, which does not include the sequence encoded by exon 6, was the dominant isoform transcribed; at western blot analysis PKP2A was the only clearly detectable isoform in all human heart samples analysed (from six different controls and the proband). Moreover, in the proband's sample, p.Arg490Trp was not associated with aberrant exon 6 splicing or mutant mRNA downregulation. Finally, a heterozygous missense variant (p.Glu2343Lys) in Desmoplakin was identified in this proband and is likely to be the disease-causing mutation. CONCLUSION: PKP2A was shown to be the major isoform expressed in human heart tissue and PKP2B protein was undetectable. The results strongly suggest that p.Arg490Trp and other variants located in PKP2 exon 6 may not be disease causing. Variant splicing also has important consequences for the interpretation of mutation analysis and genetic counselling in ARVC and other hereditary cardiac diseases.

The microscopic (optical and SEM) examination of putrefaction fluid deposits (PFD). Potential interest in forensic anthropology.

This article describes the potential interest in physical and forensic anthropology of the microscopic analysis of residues of putrefaction fluid, a calcified deposit frequently found associated with bone rests. Its sampling and analysis seem straightforward and relatively reproducible. Samples came from archeological material (Monterenzio Vecchia, an Etruscan necropolis from the north of Italy dated between the fifth and third century B.C.; body rests of Agnès Sorel, royal mistress died in 1450 A.D.; skull and grave of French King Louis the XI and Charlotte of Savoy dated from 1483 A.D.). All samples were studied by direct optical microscope and scanning electron microscopy. Many cytological, histological, and elemental analysis were possible, producing precious data for the identification of these remains and, in some cases, the cause of death.

Frequency of traumatic lesions alleged by victims of assault during police custody.

A retrospective study was undertaken to determine the frequency of traumatic lesions found in individuals alleging police brutality during official custody in the département of Hauts-de-Seine, located in the west suburbs of Paris. All medical certificates relating to the examination of 11,653 individuals detained during the year 2004 were analysed. From this population, there were 119 cases where victimized individuals alleged police assault and 245 revealed aggressive police manhandling, as indicated by traces of tight handcuffs. Among the individuals alleging police violence, most of them showed recent traumatic lesions (n=91). The majority of lesions were superficial contusions frequently located in the cervico-cephalic area. All traumatic lesions were compatible with the allegations of police assault. Neurological complications secondary to the application of handcuffs were encountered in less than 2% of the cases. Although no death was recorded in police custody during the period of the study, approximately 5% of the population that had encountered some form of police violence was found to require emergency hospitalization.

The international criminal tribunal for the former Yugoslavia (ICTY) and the forensic pathologist: ethical considerations.

Since 1991, war crimes in the former Yugoslavia have been the subject of several international medico-legal investigations of mass graves within the framework of inquiries led by the International Criminal Tribunal for the former Yugoslavia (ICTY). Forensic pathologists involved in the ICTY missions could be subjected to ethical tensions due to the difficulties of the missions, the emergent conflicts between forensic scientists of the investigating teams and the original nature of the ICTY proceedings. In order to study the nature of such ethical tensions, we sent a questionnaire to 65 forensic pathologists who have been involved in the ICTY missions. The rate of response was 38%. The majority of forensic pathologists questioned (n=18) did not know how the medico-legal data was exploited by the ICTY. Three of them have been subjected to pressures. Three of them were aware of mass grave sites knowingly not investigated by the ICTY. Fifteen considered that the ICTY respected the elementary rules of the law and four of them questioned the impartiality of the justice led by the ICTY. Two conflicting types of ethics can be drawn from these results: conviction ethics, which are shared by most of the forensic pathologists questioned, and responsibility ethics. In the former, the forensic pathologist completely agrees with the need for an international war crimes tribunal, even if such justice can be challenged regarding the respect of human rights and impartiality. In the latter, he or she needs to conduct him or herself in ways that do not infringe impartiality. As medical deontology duty requires impartiality ethics, discursive ethics are needed to ease ethical tensions and to suggest ethical guidelines. Alternatives to international justice, through a truth and reconciliation commission and by way of humanitarian missions combining victims' identification with forensic investigations for historical purposes, could be considered.

Analysis of 155 consecutive forensic exhumations with emphasis on undetected homicides.

A total of 155 consecutive forensic exhumations performed in Münster, Germany from 1967 to 2001 were evaluated retrospectively on the basis of the autopsy report, the police report and the death certificate. Histology and toxicology were performed in most cases. The postmortem intervals varied from 8 days to 8 years. Compared to other countries, the autopsy rate was low (1.2-1.4%) and the exhumation rate high (0.016%): principle of reciprocity. The cause of death could be clearly determined in 103 cases (66.5%) and histology or toxicology were decisive in 40%. Some findings were discernable using immunohistochemistry after considerable postmortem intervals, such as acute myocardial infarction after 1 year and pneumonia after 2 years and a diazepam intoxication was determined after 4.5 years. Major deviations between the cause of death as stated on the death certificate and as diagnosed after autopsy existed in 57 cases (37%). A more detailed analysis revealed five subgroups. 1. primary suspicion of intoxication (n=18) confirmed in 6 cases including 3 homicides (with parathion, clozapin, diazepam) which are described in more detail. 2. primary suspicion of homicide other than poisoning (n=51) confirmed in 19 cases. There was a serial killing of 15 patients by injection of air. In the remaining 4 cases, a shaken infant, craniocerebral injuries from blows with beer bottles, a craniocerebral gunshot and a multiplicity of blunt force injuries were diagnosed. The latter two cases are described in more detail. Superficial external examinations and the low autopsy rate were 2 common reasons for the occurrence of "buried homicides" (n=22)-not a single forensic autopsy had been performed directly after the death of the victims. 3. primary suspicion of medical malpractice (n=39). 4. accidents including traffic accidents (n=30). 5. clarification of the cause of death, circumstances or identity (n=17). Exhumations were frequently successful for recovering evidence which should better have been collected immediately after the death of an individual. Exhumations can also be regarded as an instrument to evaluate the quality of death certificates and death investigations.

Myocarditis associated with Hashimoto's disease: a case report.

We report on a sudden cardiac death case involving a 40-year-old man with no known medical history. Forensic autopsy showed lymphocytic myocarditis associated with lymphocytic thyroiditis. In both the heart and the thyroid gland, the inflammatory foci often had a nodular pattern with a germinal centre. Virological and toxicological analyses were negative. Postmortem biochemistry showed a slight increase in TSH in combination with normal T3 and T4 blood levels suggesting hypothyroidism. High titres of antiperoxidase and antithyroglobulin antibodies with normal levels of TSH receptor antibodies, in addition to biological hypothyroidism and lymphocytic inflammation were consistent with the diagnosis of Hashimoto's thyroiditis. Immunohistochemical studies excluded a lymphoma and showed no evidence of viral myocarditis. In contrast to Grave's disease, Hashimoto's thyroiditis has never been reported in association with myocarditis as a cause of sudden death. We conclude that the cardiac immunological and histological pattern, similar to that found in the thyroid gland suggests an autoimmune myocarditis.

Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa.

Identification of spermatozoa is the biological evidence most often sought in specimens from rape victims. Absence of spermatozoa usually terminates biological investigations, and the victim's testimony can be contested. We assessed the utility and reliability of PCR amplification using Y-chromosomal STR polymorphisms in specimens from female victims of sexual assault with negative cytology. One hundred and four swabs without spermatozoa detected by cytology were collected from 79 alleged sexually assaulted female victims and amplification of Y-STR and of amelogenin was performed.Overall, Y-chromosome was detected and evidenced sexual penetration in 28.8% of swabs. In the population of victims examined more than 48 h after the sexual assault, Y-STR were still evidenced in 30% of the cases. These results show that swabs should be taken from victims for Y-chromosome DNA typing even after long delays between sexual assault and medical examination.

Assessment of right ventricular lipomatosis by histomorphometry in control adult autopsy cases.

A histomorphometry study was carried out to assess the degree of right ventricular lipomatosis in control autopsy cases and to evaluate if this was correlated with parameters such as sex, age, body mass index (BMI) and heart weight. A total of 70 adult cases were selected from cases of violent death between 1991 and 1999 and where autopsies were carried out in the Department of Pathology and Forensic Medicine in Garches. All cases with heart pathology, abnormal BMI or putrefaction were excluded. Cases with lung or liver pathology were also excluded. Furthermore, 10 adult autopsy cases who died suddenly of arrhythmogenic right ventricular cardiomyopathy (ARVC) were compared with 10 age and sex-matched control cases. Details on sex, age, BMI and heart weight were obtained from the post-mortem records. For each case one sample of the right front ventricular wall was fixed in 10% neutral saline-buffered formalin and one 5-microm-section was stained with haematoxylin and eosin. The Leica Quantimet 500 analysis system was used for the histomorphometrical study. The mean degree of lipomatosis was measured under blind conditions in the ventricular wall and epicardial fat was excluded. Covariance analysis and the Wilcoxon test were used for statistics. The mean age of the control population was 37.5 years, the sex ratio was 1.9:1 (male:female). The mean degree of lipomatosis was 17.03% and the degree of lipomatosis was significantly correlated with age (p = 0.0029) but not with sex, BMI and heart weight. There was a statistically significant increase in fat in ARVC cases compared with age and sex-matched controls (p < 0.001). Fat infiltration of the right ventricle could be an adipose involution due to an ageing process and heavy fat infiltration can be difficult to distinguish from ARVC. Our study suggests that fat infiltration is not essential for the post-mortem diagnosis of ARVC which also requires fibrosis and degenerating myocytes trapped within areas of fibrosis.

[Hygiene, architecture and autopsy]. / Hygiène, architecture et autopsie.

The 24(th) August 1998 decree related to technical prescriptions applicable to hospital mortuaries, to be enacted as of September 11(th) 2001, compels hospital administrations and involved personnel to consider architectural concepts of autopsy room. Nevertheless, the technical prescriptions of this decree seem to be inadequate because an autopsy room requires specific functional and technical organization such as adequate flooring, adapted air-conditioning system and plumbing and of course appropriate autopsy tables. Due to imminent enactement of accreditation procedures, it is likely that in the near future, autopsies will only be performed in centres combining scientific competence and sanitary requirements.

Neuropathology and neurodegeneration in human immunodeficiency virus infection. Pathogenesis of HIV-induced lesions of the brain, correlations with HIV-associated disorders and modifications according to treatments.

A variety of HIV-induced lesions of the central nervous system (CNS) have been described, including HIV encephalitis, HIV leukoencephalopathy, axonal damage, and diffuse poliodystrophy with neuronal loss of variable severity resulting, at least partly, from an apoptotic process. However, no correlation could be established between these changes and HIV dementia (HIVD). From our study of HIV infected patients, it appeared that neuronal apoptosis is probably not related to a single cause. Microglial and glial activation, directly or indirectly related to HIV infection, plays a major role in neuronal apoptosis possibly through the mediation of oxidative stress. In our patients with full-blown AIDS, this mechanism predominated in the basal ganglia and correlated well with HIVD. Axonal damage, either secondary to microglial activation, or to systemic factors also contributes to neuronal apoptosis. Although massive neuronal loss may be responsible for HIVD in occasional cases, we conclude that neuronal apoptosis is a late event and does not represent the main pathological substrate of HIVD. The dementia more likely reflects a specific neuronal dysfunction resulting from the combined effects of several mechanisms, some of which may be reversible. Introduction of highly active antiretroviral therapy dramatically improved patient survival, however, its impact on the incidence and course of HIVD remains debatable. In our series, the incidence of HIVE has dramatically decreased since the introduction of multitherapies, but a number of cases remain whose cognitive disorders persist, despite HAART. The poor CNS penetration of many antiretroviral agents is a possible explanation, but irreversible "burnt out" HIV-induced CNS changes may also be responsible.

Symmetrical necrosis of the solitary tract nuclei as a contributory cause of death.

A 64-year-old man died in spite of surgery 4 days after attempting suicide. He first tried to hang himself with a rope and when the hanging did not succeed, he cut his throat with a knife. The autopsy showed four sutured cervical wounds with laryngeal wounds but without associated important vascular injury. The neuropathological study revealed two watershed-type haemorrhagic infarcts, involving the left occipital lobe and the left cerebellum. It also showed a symmetrical necrosis of solitary tract nuclei in the medullary tegmentum. Such a lesion is likely to result from sudden acute transient circulatory failure and might have played a role in the secondary autonomous cardiac and respiratory dysfunctions following a non-lethal trauma.

Progressive multifocal leukoencephalopathy and oligodendroglioma in a monkey co-infected by simian immunodeficiency virus and simian virus 40.

A rhesus monkey experimentally inoculated with simian immunodeficiency virus (SIV) mac251 was killed 42 months later because of poor general condition. CD4 lymphocyte count which was 3,430/mm3 before inoculation, had decreased to 638/mm3 2 months before death. Neuropathological examination revealed changes characteristic of progressive multifocal leukoencephalopathy (PML) in the white matter of the cerebral hemispheres and brain stem. In situ hybridization was negative for JC virus but markedly positive for simian virus 40 (SV40) in the nuclei of many oligodendrocytes. Many oligodendrocytes also expressed p53. Within an area involved by PML, there was a densely cellular tumor with honeycomb appearance and elongated vessels characteristic of oligodendrogliomas. Within the tumor in situ hybridization for SV40 and immunocytochemistry for p53 were negative. Opportunistic infection by SV40 has been occasionally reported in experimentally SIV-infected monkeys resulting in PML or malignant astrocytoma. Association of JC virus-induced PML and astrocytomas has been reported in three human cases without AIDS. In those cases, as in our monkey, polyomaviruses (SV40 or JC virus) were expressed in the areas with PML but not in the glial tumor. Association of PML and oligodendroglioma has not been reported previously to our knowledge. The relationship between oligodendrocyte proliferation and polyomavirus infection of oligodendrocytes is unclear. Our findings suggest that binding of the viral protein to p53 may result in inactivation of the pro-apoptotic protein favoring the proliferation of a randomly occurring tumoral clone of oligodendrocytes.

Increase of pulmonary density of macrophages in sudden infant death syndrome.

A 1996 cytodensitometric study found increased cellular density in the pulmonary parenchyma of infants who died of sudden infant death syndrome (SIDS). The present study clarifies these results in quantifying the density of immunohistochemical subtyped inflammatory cells. Histomorphometry was used to compare the density of macrophages, granulocytes and T and B lymphocytes in the lungs of two groups of infants. From the post-mortem records of infant deaths between 1983 and 1995, 29 (mean age = 5 months) were randomly selected including 16 cases of SIDS and 13 who died of other non-pulmonary causes. Densities of immunoreactive cells were measured under blind conditions in the parenchyma. The mean density of macrophages was significantly higher in cases of SIDS compared with the controls (P = 0.0318), but there were no differences for the lymphocytes and the granulocytes. These morphometrical results must be interpreted within the methodological limits of this study, especially the non-uniform level of lung inflation between selected subjects. However, the differences in level of inflation are not sufficient to explain the observed increase of macrophage density. Indeed, the mean values of alveolar surface area, which represent an indirect measure of lung inflation, are not significantly different between the two groups. Increase of pulmonary macrophage density in SIDS agrees with three non-exclusive hypotheses: (1) an abnormal inflammatory reaction by expression of Th1 helper cell phenotype activation; (2) consequence of passive smoking; and (3) post-agonal mechanisms. Bacterial superantigens produced by toxigenic bacteria in the respiratory tract could play a role as a trigger factor that initiates a fatal cascade with overproduction of cytokines leading to death. The significant increase of pulmonary macrophage density would be the morphological expression of this potential mechanism of death.